1. Field of the Invention
The invention is in the field of organic chemistry. The invention relates in part to a process for conversion of (3R,3xe2x80x2R,6xe2x80x2R)-lutein to (3R,3xe2x80x2S,6xe2x80x2R)-lutein (3xe2x80x2-Epilutein), a carotenoid precursor for industrial production of naturally occurring (3R,3xe2x80x2R)-zeaxanthin.
2. Related Art
As a result of a high intake of fruits and vegetables, 34 carotenoids and their metabolites are found in human serum and tissues at varying concentrations. Among these, only two dietary carotenoids, (3R,3xe2x80x2R,6xe2x80x2R)-lutein and (3R,3xe2x80x2R)-zeaxanthin, accumulate in the human retina. Epidemiological and experimental data suggest that the function of these carotenoids is to protect the photo-sensing cells in the human retina, and particularly macula, from exposure to short-wavelength blue light, and thus prevent age-related macular degeneration (AMD) (Bone, R. A., et al., Invest. Ophthalmol. Vis. Sci. 34:2033-2040 (1993); Seddon, J. M., et al., J. Am. Med. Assoc. 272:1413-1420 (1994)).
While several patented processes for the industrial production of (3R,3xe2x80x2R,6xe2x80x2R)-lutein have been reported, (3R,3xe2x80x2R)-zeaxanthin is not commercially available. Currently the most promising route to this carotenoid is by chemical synthesis (Widmer et al., Helv. Chim. Acta 73:861-867 (1990)). However, the production of (3R,3xe2x80x2R)-zeaxanthin by total synthesis can be quite costly. Furthermore, the absence of possible residual contaminants in (3R,3xe2x80x2R) zeaxanthin prepared by synthesis must be established before this carotenoid can be safely used as a nutritional supplement or food coloring additive.
U.S. Pat. No. 5,523,494, European Patent Appl. 834536 and WO99/03830 describe the conversion of commercially available (3R,3xe2x80x2R,6xe2x80x2R) lutein or crude marigold meal to (3R,3S,meso)-zeaxanthin by base-catalyzed isomerization. (3R,3S,meso)-Zeaxanthin is absent from foods and its stereochemistry is different from that of dietary (3R,3xe2x80x2R)-zeaxanthin.
WO97/31894 describes a process wherein (3R,3xe2x80x2R,6xe2x80x2R)-lutein is first converted to (3R,3S,meso)-zeaxanthin and then is oxidized to xcex2,xcex2-carotene-3,3xe2x80x2-dione. In the final step of this process xcex2,xcex2-carotene-3,3xe2x80x2-dione is reduced with sodium or potassium borohydride to give a racemic mixture of (3R,3xe2x80x2R)-zeaxanthin, (3R,3xe2x80x2S,meso)-zeaxanthin, and (3S,3xe2x80x2S)-zeaxanthin (Scheme 1). 
Therefore with the exception of total synthesis, the other reported procedures either prepare (3R,3xe2x80x2S,meso)-zeaxanthin, which is not the natural dietary form of this carotenoid, or use chemical reagents and additional steps to prepare a racemic mixture of (3R,3xe2x80x2R)-zeaxanthin, (3R,3xe2x80x2S,meso)-zeaxanthin, and (3S,3xe2x80x2S)-zeaxanthin.
The present invention seeks to produce a precursor to (3R,3xe2x80x2R)-zeaxanthin in high diastereomeric excess (de). The precursor may then be transformed to (3R,3xe2x80x2R)-zeaxanthin by methods that are well known in the an.
According to the present invention, (3R,3xe2x80x2R,6xe2x80x2R)-lutein can be epimerized at C-3xe2x80x2 in one convenient step using dilute acids to give a 1:1 diastereomeric mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein as shown in Scheme 2. 
In the course of the work-up of the epimerization reaction, most of the (3R,3xe2x80x2R,6xe2x80x2R)-lutein is removed from this 1:1 mixture by filtration to produce a diastereomeric mixture in which the ratio of 3xe2x80x2-epilutein to (3R,3xe2x80x2R,6xe2x80x2R)-lutein is in the range of 3.4 to 3.5. A de of 54-90% can be accomplished by a) solvent extraction, b) preferential crystallization, c) Soxhlet extraction, d) enzymatic acylation, and e) supercritical extraction with carbon dioxide of either an about 1:1 mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein or the partially separated mixture (with ratio of 3.4 to 3.5).
Only enzymatic acylation of the 1:1 diastereomeric mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein, affords 3xe2x80x2-epilutein in 90% de whereas the other methods require additional purification to increase de of this carotenoid. The partially separated mixture can be subjected to low temperature crystallization to crystallize most of the (3R,3xe2x80x2R,6xe2x80x2R)-lutein and increase the de of 3xe2x80x2-epilutein in the mother liquor of this crystallization. Under these conditions, most of the (3R,3xe2x80x2R,6xe2x80x2R)-lutein is crystallized and can be recovered. At any stage of these processes, the recovered solid which is predominantly enriched in (3R,3xe2x80x2R,6xe2x80x2R)-lutein, can be recycled into the epimerization reaction.
The isolated 3xe2x80x2-epilutein may then be directly converted to naturally occurring (3R,3xe2x80x2R)-zeaxanthin by base-catalyzed isomerization (vide supra). All of the above separation procedures can be readily implemented on industrial scale with the advantage that the recovered (3R,3xe2x80x2R,6xe2x80x2R)-lutein can be recycled and epimerized to 3xe2x80x2-epilutein. The present invention does not employ any reagents other than commonly used organic solvents and acids which can be safety handled under mild conditions.
In particular the invention relates to a method of epimerizing (3R,3xe2x80x2R,6xe2x80x2R)-lutein to give a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein, comprising reacting (3R,3xe2x80x2R,6xe2x80x2R)-lutein in the presence of aqueous acid in a water miscible organic solvent to give a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein.
The invention further relates to a method of purifying 3xe2x80x2-epilutein from a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein comprising extracting the mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein with an organic solvent and recovering the 3xe2x80x2-epilutein from the organic solvent.
The invention further relates to a method of purifying 3xe2x80x2-epilutein from a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein comprising low temperature crystallization of the mixture in a C1-4 alcohol and recovering the 3xe2x80x2-epilutein from the alcohol.
The invention further relates to a method of purifying 3xe2x80x2-epilutein from a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein comprising:
(a) reacting a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein with an acyl donor in the presence of lipase PS from Pseudomonas cepacia or lipase AK from Pseudomonas fluorescens in an first organic solvent;
(b) adding a second organic solvent to dissolve the mixture and removing the enzyme by filtration to give a filtrate;
(c) concentrating the filtrate to give a residue;
(d) adding a C5-7 hydrocarbon or ether to the residue to give a solution in which 3xe2x80x2-epilutein and 3xe2x80x2-epilutein-3xe2x80x2-acetate are preferentially solubilized;
(e) filtering the solution to give a filtrate;
(f) hydrolyzing the 3xe2x80x2-epilutein-3xe2x80x2-acetate contained in the filtrate to give 3xe2x80x2-epilutein; and
(g) recovering the 3xe2x80x2-epilutein;
thereby obtaining purified 3xe2x80x2-epilutein.
The invention further relates to a method of purifying 3xe2x80x2-epilutein from a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein comprising extracting the mixture with supercritical carbon dioxide and evaporating the carbon dioxide to give purified 3xe2x80x2-epilutein.
The invention further relates to a method for producing 3xe2x80x2-epilutein comprising:
(a) epimerizing (3R,3xe2x80x2R,6xe2x80x2R)-lutein to 3xe2x80x2-epilutein in the presence of an aqueous acid in a water miscible organic solvent thereby giving a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein; and
(b) separating 3xe2x80x2-epilutein from (3R,3xe2x80x2R,6xe2x80x2R)-lutein.
The invention further relates to a method for converting (3R,3xe2x80x2R,6xe2x80x2R)-lutein to a mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein comprising:
(a) reacting (3R,3xe2x80x2R,6xe2x80x2R)-lutein with an aqueous acid in a solvent at ambient temperature to obtain a crude product;
(b) neutralizing the crude product with aqueous base; and
(c) removing the water by partitioning the crude product into a water immiscible organic solvent;
thus obtaining a crude diastereomeric crystalline mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein.
The invention further relates to a method for the separation of 3xe2x80x2-epilutein from a mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein by enzymatic acylation, comprising reacting 3xe2x80x2-epilutein with an acyl donor in the presence of a lipase in pentane, hexane or TBME at 36xc2x0 C. to convert 95% of 3xe2x80x2-epilutein to 3xe2x80x2-epilutein-3xe2x80x2-acetate while (3R,3xe2x80x2R,6xe2x80x2R)-lutein remains unreacted; subjecting the resulting 3xe2x80x2-epilutein-3xe2x80x2-acetate to hydrolysis with alcoholic potassium or sodium hydroxide at ambient temperature; removing the base by extracting the product with water and an organic solvent; and evaporating the solvent to obtain diastereomeric luteins comprising 95% 3xe2x80x2-epilutein and 5% (3R,3xe2x80x2R,6xe2x80x2R)-lutein as red crystals.
The invention further relates to a method of preparing 3xe2x80x2-epilutein-3xe2x80x2-acetate comprising reacting a mixture of (3R,3xe2x80x2R,6xe2x80x2R)-lutein and 3xe2x80x2-epilutein with an acyl donor in the presence of lipase PS from Pseudomonas cepacia or lipase AK from Pseudomonas fluorescens. 
The invention further relates to a method for partial separation of 3xe2x80x2-epilutein from a diastereomeric mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein obtained according to the present invention by extracting the crude crystalline mixture with pentane or petroleum ether (b.p.=35-60xc2x0 C.) in a Soxhlet apparatus to obtain a pentane or petroleum ether soluble fraction comprising of a diastereomeric mixture of 3xe2x80x2-epilutein (80%) and (3R,3xe2x80x2R,6xe2x80x2R)-lutein (20%), evaporating the solvents to obtain a crystalline mixture enriched in 3xe2x80x2-epilutein.
The invention further relates to a method for separating 3xe2x80x2-epilutein from a diastereomeric mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein comprising extracting the diastereomeric mixture of luteins with carbon dioxide thereby extracting most of 3xe2x80x2-epilutein with carbon dioxide and leaving behind most of the (3R,3xe2x80x2R,6xe2x80x2R)-lutein thus yielding a product consisting of 86% 3xe2x80x2-epilutein and 14% (3R,3xe2x80x2R,6xe2x80x2R)-lutein.
The invention further relates to a method for separating 3xe2x80x2-epilutein from a diastereomeric mixture of 3xe2x80x2-epilutein and (3R,3xe2x80x2R,6xe2x80x2R)-lutein comprising low temperature crystallization with an alcohol at xe2x88x9270xc2x0 C. to crystallize most of the (3R,3xe2x80x2R,6xe2x80x2R)-lutein and increasing the purity of 3xe2x80x2-epilutein in the mother liquor of this crystallization; and evaporation of the alcohol; thus obtaining red crystals containing 94% 3xe2x80x2-epilutein and 6% (3R,3xe2x80x2R,6xe2x80x2R)-lutein.